Clinical chemistry is a field concerned, inter alia, with the analysis of various substances to determine elements, or "analytes" thereof. Of particular importance in this field is the analysis of samples taken from humans in order to diagnose for diseases, to determine licit or illicit drug consumption and overdoses, and so forth. Generally, the analytical techniques employed to carry out these processes involve analyzing a sample of a body fluid, such as urine, blood, feces, and other biological exudates. Among the analytes determined in various analytical tests are glucose levels (in blood or urine); bilirubin (blood); hormones, such as human chorionic gonadotropin, or hCG (urine), which leads to diagnosis or monitoring of conditions such as diabetes. Measurement of hCG is a common way of determining whether a woman is or not pregnant. Opiates such as morphine and heroin, and toxins such as digoxin are also determined in standard analytical assays, as are essential ions, such as Ca.sup.2+, and K.sup.+. Examples of the art as applied to determination of specific analytes include Sena, et al., Clin. Chem. 34(3): 594 (1988) (creatinine); Hafkenscheid, et al., Clin. Chem. 34(1): 155-157 (1988) (gamma glutamyl-transferase, creatine kinase, etc.); U.S. Pat. No. 4,069,016 (bilirubin); U.S. Pat. No. 4,452,887 (glucose); Reissue 32,016 (lactic acid and lactase). These references are merely exemplary, as even the most cursory of reviews will produce many different assay methods for various analytes.
Assays for determining sample analytes can be divided into two major groups: homogeneous and heterogeneous assays. Of the two, it is the latter group to which this invention is directed, and the following discussion concerns this group.
A heterogeneous assay involves two phases: solid and liquid. The liquid phase contains the analyte to be determined, and the liquid is contacted to a solid phase, which has been pretreated to contain some substance which reacts with the analyte to be determined, producing some observable reaction.
The field contains innumerable examples of heterogeneous assays, and the observable reactions produced therein. Exemplary of these are U.S. Pat. No. 4,376,110, which describes so-called "sandwich" assays. In these assays, a sample is contacted to a solid phase containing an antibody to the analyte being determined. Binding occurs between the two, and then a second monoclonal antibody, containing a label is contacted to the bound complex. The label, which can be, e.g., an enzyme is then "read" by contacting it with a substrate, forming a detectable color. Measurement of the intensity of the color is a measure of how much analyte is bound to the solid phase. The color can be read visually, or by various photometric or spectrophotometric means, which are well known to the skilled artisan.
More typical of heterogeneous assays, however, are those described by e.g., U.S. Pat. No. 4,069,016 (Wu), cited supra. In this patent, the bilirubin assay involves contacting the sample to a solid phase, which contains various chemical substances which interact in the presence of bilirubin to produce a detectable shift in the color of the sample. This shift in color is read, as indicated supra, as an indication and measurement of bilirubin content. Also typical of such systems is U.S. Pat. No. 4,452,887, also cited supra. The device discussed therein is adapted for measuring glucose. In the device, referred to as a "dry type" apparatus, glucose is acted upon by glucose oxidase, forming a product which is in turn acted upon by other reactants, leading to the production of hydrogen peroxide. The hydrogen peroxide, in turn, reacts with an indicator such as a tetrazolium salt (e.g., 3,3',5,5' tetramethylbenzidine or "TMB"), or a different nitro- or phenyl type indicator, forming a color. One then observes or measures the color as an indication of the presence and/or amount of glucose. This serves as a means for diagnosing, e.g., diabetes mellitus, or to monitor blood sugar levels of a diabetic.
Various formats have been developed in the field for performing assays of the type described supra. One kind of strip, the "impregnated bibulous paper" type, is described, e.g., in U.S. Pat. Nos. 4,446,232; 4,235,604; and 4,459,358. All of these devices rely on the diffusion of liquid through an absorbent material, such as filter paper. If the liquid contains the analyte of interest, various reactions occur at reaction stations, or "zones" spaced throughout the device. The idea of reactions occurring at different points in the apparatus can also be a feature of the "layered" type of apparatus, represented, e.g., by U.S. Pat. Nos. 4,069,015; 3,992,158; 4,256;693; 3,901,657; and 4,144,306. In these devices, different layers are either connected, or are in fluid contact with each other such that clear demarcations can be seen in the different zones of the devices. These zones serve different functions, including evenly distributing the analyte containing sample for distribution through the device (a "spreading layer", e.g., in U.S. Pat. No. 3,992,158); and reagent zones, whose function is evident, as well as light impermeable layers. This feature serves to present a more easily readable signal after color formation takes place, because stray light does not interfere with the generated signal.
Yet another configuration of analytical test devices is the type which, physically, most represents the idea of a "test strip". These devices combine features of the layered device with those of the bibulous paper device, sand are represented, e.g., by U.S. Pat. Nos. 4,477,575, and 4,076,502. In these devices a sample is applied to a region of the strip which is configured in a layer fashion, and passes therethrough, and reaches a region which is configured in a fashion similar to bibulous paper strips. Frequently, in the first part of the device the sample undergoes preparatory steps, such as buffering or filtering, and undergoes chemical analysis in the second portion.
Yet another version of a test device has recently become available, the so-called "channel model" test strip. This type of device presents a sealed, canal shaped device through which sample flows, and is positioned with various reaction sites where sample analysis takes place. Attention is drawn to German patent specification DE 3 643 516 which corresponds to U.S. Pat. Application Ser. No. 134,950.
All of these devices can be used for analysis of some body fluids. A problem arises, however, with the analysis of whole blood, which requires special adaptation of the test device.
It will be recognized that whole blood has a distinct, dark red color. This property of blood makes it extremely difficult to analyze it in systems where color formation or change is involved, because the red color of the blood obscures or interferes with the indicator reaction going on.
In recognition of this problem, various test devices have been developed which do permit one to analyze whole blood. These devices usually include a feature which selectively filters erythrocytes from the sample, permitting passage of clear plasma into the actual testing region. Thus, U.S. Pat. No. 4,477,575 features a filtering membrane of glass fibers of particular dimensions. U.S. Pat. No. 4,256,693 suggests that filter paper can be used to remove the red blood cells, while U.S. Pat. No. 4,476,222 proposes coating a carrier with a mixture of polymethacrylate and polyvinyl formal. U.S. Pat. No. 4,594,327 incorporates a ligand receptor specifically chosen to bind to, and remove erythrocytes from the sample, while U.S. Pat. No. 4,478,944 teaches specific polymeric coatings which impede passage of erythrocytes into a testing region. Other test devices feature membrane filters (U.S. Pat. No. 3,663,374) or the use of carbohyrdate or amino acid molecules to remove the erythrocytes from the sample. See, e.g., U.S. Pat. Nos. 4,678,757 and 3,552,958. U.S. Pat. No. 4,543,338, also teaches such a device, where a test paper is coated with a partially cross-linked polymer. As this last patent's title attests, these are all "Wipe-off Test Devices", in that they require the investigator to wipe away the filtered erythrocytes from the test system.
As pointed out, the foregoing devices all require wiping off of the filtered red blood cells. This presents various problems both to the clinician and the layperson, from the viewpoint of accuracy, convenience and of safety. Blood samples can often contain infectious agents or toxins which present a hazard to the individual analyzing the sample. One contemporary example of this human immunodeficiency virus (HIV), which is known to be transmissible via the blood. It is of course desirable to alleviate or eliminate the problem of putting the investigator into contact with the blood sample.
From the standpoint of accuracy, test devices are extremely sensitive and delicate equipment. As such, elimination of as much intervention as possible is a desired goal. Finally, in terms of convenience, it will be understood that while the layperson might perform a single test per day in, e.g., home monitoring of glucose levels, the laboratory clinician may routinely perform hundreds of such tests in a day. The "wiping-off" which may take only a few seconds or a minute for one test, is magnified by the sheer number of tests which must be carried out in a given time period.
It will be seen, then, that one is confronted with an apparently resolvable problem. If one the one hand, the red blood cells are not removed from a sample, analytical sensitivity is impaired. If, on the other hand, the red blood cells are removed, other problems arise. Separating the red blood cells from the sample by, e.g., centrifugation prior to analysis, is not an option for the home tester, and presents problems such as erythrocyte lysis, etc., in the laboratory.
A key advance in the field was presented by the so-called "channel model" device disclosed in German Patent Specification 36 43 516, corresponding to U.S. Ser. No. 134,950, of which the present inventor is a co-inventor.
It was realized in the course of development of test devices for analyzing whole blood that there were three areas of major concern. The device had to facilitate flow of blood therein, enable it to be drawn out, and had to show complete removal of the blood sample. All three criteria had to be satisfied in order to achieve any meaningful improvement.
The "channel model", referred to supra, accomplished this to some extent. Its structure allows for improved blood flow into the capillary space it provides, and achieves rapid withdrawal or drainage. This model, however, did not always solve the problem of complete withdrawal. Unless an airtight seal is achieved in the channel model, when blood flows in, air is drawn in as well. Air creates "bubbles", which break up the continuous stream of blood in the device. The result is that blood further along the device than the air bubble is removed, but with the break in continuity, the blood behind the bubble is not. The sample becomes discontinuous, and blood backs up in the device.
Additionally, the channel model construction requires an amount of sample which is rather large and inconvenient, especially in "user friendly" or "at home" type tests. In these, the consumer/user must prick a finger, e.g., and hold it to the device until sufficient blood is taken up for analysis.
It is an object of this invention to present an apparatus useful in determining an analyte in a fluid sample, such as whole blood, which is not subject to the above identified problems, which requires less sample than other available devices, which can carry out analyses more quickly than other available systems, and which delivers blood to a reaction area quickly, followed by rapid removal.
It is a further object of of the invention to provide a method for analyzing a fluid sample for an analyte using the aforementioned device.
How these and other objects of the invention are achieved will be seen from review of the disclosure which follows.